PCS124 and PCS069 protease mutants
5.4 Discussion
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A431V model. Additionally, several interactions were observed with A431V, namely, multiple alkyl bonds with WT residues V32 and I47 in PR. Finally, whilst D25 did not hydrogen bond with the mutated NC|p1 substrate, it formed close vdW interactions to I47 in PR which in turn was closely bonded to A431V in Gag.
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this suggests that the same mechanism employed to evade drug binding is used to maintain substrate cleavage. Specifically, as previously discussed, changing the AA torsion angles can alter protein conformity (Thukral et al., 2007; Ponnuraj and Saravanan, 2017; Saravanan and Selvaraj, 2017). Additionally, the level of asymmetry in the PCS069 model indicates that each chain is rapidly regulated either to constrict or flex to allow movement for the highly rotatable, bulky Gag CS. Therefore, it is conceivable that the asymmetrical AA fluctuations in a homodimeric protein may evade drug binding through the rapid regulation of its subunits.
While it is known that Gag cleavage occurs over several sequential steps as discussed in chapter one, the exact structural mechanism for this process is debatable (Mattei et al., 2018). Even though the PIs were designed to out-compete the natural substrate (Spearman, 2016), the HIV-1 Gag, consisting of approximately 500 AAs (Fun et al., 2012), is a large protein in nature. Therefore, it is also likely that the 10 bulky AAs recognized by PR for cleavage is also molecularly large.
Separate studies conducted by Prabu-Jeyabalan et al. (2002) and King et al. (2004) showed that the PR’s substrates occupies a consensus volume within the binding cavity. Ӧzen et al. (2011) hypothesized that the ability of the CSs to fit within the substrate envelope is also determined by the substrate size and dynamics. This suggests that PR’s capability to incorporate Gag essentially depends on the peptide’s rotamers and ultimately its flexibility. Furthermore, the G435 and K436 residues contributes to the fundamentally flexible nature of the NC|p1 substrate (Ӧzen et al., 2012).
Additionally, this flexibility may also contribute to the stronger binding observed in the PCS- A431V complexes as compared to the drug-bound PR models. The increased binding affinity for the NC|p1 CS also reiterates that the PRMs were compatibly selected to accommodate Gag.
Moreover, the 12 kcal/mol difference between the PCS124 and PCS069 models indicates a selective advantage to acquiring a 4th major PRM in favour of three (M46I+I54V+V82A).
Therefore, it is accepted that via alternate mechanisms, patterns of mutations act synergistically to evade drugs (Agniswamy et al., 2016).
Lastly, to explore the intricate interactions between the mutated Gag and PR, 2D interaction maps were constructed. Our analyses revealed that key hydrogen bonds involving several Gag residues (E428, Q430, N432, L434 and G435) across the substrate was observed in the PCS124-A431V model. While substrate dynamical flexibility allows for its incorporation (Ӧzen et al., 2011), this suggests that the formation of hydrogen bonds arose to stabilize the CS, keeping it relatively static for cleavage to occur. Furthermore, our analyses also revealed an important hydrogen bond with
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PR’s catalytic D25 and the carbon atom of isoleucine at position 437 in the NC|p1 Gag CS. This bond suggests that the CS rotated into and downwards of PR’s substrate cavity to catalyze the reaction. Incidentally, this could be seen Figure 5.3 where the isoleucine extended down PR. Of note, Gag Q430, G435, K436 and I437 in Gag are also sites at which drug resistance/exposure associated mutations occur (Doyon et al., 1996; Gonzalez et al., 2004; Ghosn et al., 2011; Knops et al., 2011). As such, these regions in the substrate are variable and can therefore allow for alternate substrate poses to develop when PR is unable to actively accommodate the CS.
Interestingly, the close interaction of the A431V Gag mutation with G27_PR and I47_PR indicate that the slightly larger valine substitution in place of alanine brings the active site and flaps closer together thus constricting the available space and allowing for greater binding affinity.
Furthermore, the proximity of M46I in PR and the phenylalanine of F433 in Gag suggests that coordination between these two residues can regulate movement within the flaps. Of note was the interaction of the I50’s to each other and the substrate. Tóth and Borics (2006) showed that the enzyme can exist in open, semi-open and curled conformations during a single MD simulation and that the distance between I50’s (flap tips) indicate which conformation was favoured at the time. Finally, the bond between L10F_PR and A431V_Gag which was also observed in our Gag- PR BN (chapter three), suggests that coordination between these two residues are important for substrate recognition and linkage.
The increase of hydrogen bonds between the PCS069 PR and the NC|p1 substrate suggests that the L76V mutation in PR significantly contributes toward substrate recognition and favourbale cleavage rather than evading drug binding directly. Generally, hydrogen bonds facilitate protein- ligand binding through the displacement of receptor water molecules (Chen et al., 2016). In essence, either the water molecules are displaced by the ligand or are subtly shifted (Huggins and Tidor, 2011). While water molecules are important mediators in protein-ligand binding (Brenk et al., 2006), Chen et al. (1998) showed that in an enzyme-inhibitor complex, the ligand displaced an active site water molecule which created favourable inhibitor orientation. Thus, in our study, the extensive formation of hydrogen bonds suggests that protein water displacement may have occurred to properly orient the ligand within the substrate cavity. Additionally, these data also revealed that the L76V PR mutation also closely interacted with Q430 in Gag. Incidentally, Q430_Gag was also involved in four out of the 12 hydrogen bonds. A study conducted by van Maarseveen et al. (2012) on NC|p1 CS efficiency on resistance revealed that AA position of the substrate significant correlated with the difference between the NC|p1 430–435 residues outside PR’s cavity and within the active site. Therefore, Q430 as well as A431V in Gag can alter PR’s active site dynamics for efficient substrate cleavage.
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While the hydrogen bonds between the PCS069 PR and NC|p1 CS hold the substrate in place, the CS seemingly “sits” at the top, closer to PR’s flaps (Figure 5.3). This observation was pronounced by the hydrogen bond linking A431V in Gag to G48 in PR as well as the alkyl bond between I47_PR and A431V_Gag. Studies have shown that the positioning of PR’s flaps correlated with its sensitivity to the PIs (Wlodawer et al., 1989; Yedidi et al., 2014) suggesting that the same mechanism can be employed when bound to the mutated NC|p1 substrate. Additionally, a study conducted by Khan et al. (2018) suggested that switching between flap positioning during inhibitor binding requires additional changes in PR’s conformation which may result in significant energy fluctuations. In this study, conformational changes in other parts of PR were observed by interactions between PRMs L10F, V82A and L76V (Figure 5.5; dotted black lines).
This suggests that when L76V is present together with A431V_Gag, the mutational dynamics between this PR resistance combination (L10F+M46I+I54V+V82A) changes to constrict these regions in PR and allow for efficient substrate processing in favour of drug binding. Contrastingly, the switch from hydrogen bonding to vdW interactions between D25_PR and the substrate indicates an indirect mechanism of cleavage which is consistent with studies evaluating L76V, as previously discussed (chapter four). Noteworthy, while vdW interactions are considered weak, these forces are often important in the interaction and shape of molecules (Atkins and de Paula, 2006). Therefore, in this instance, cleavage is coordinated by the flaps and strong vdW interactions rather than direct active site dynamics.
5.5 Conclusions
In summary, these data indicate that the mutated PR depicted great affinity for the NC|p1 Gag CS. Specifically, the A431V Gag mutation coordinated several PR residues to aid in substrate recognition and efficient binding. In addition, the PRMs actively work together to provide a compatible conformation that can accommodate Gag. Particularly, PRM L76V plays an important role in coordinating PR resistance dynamics, suggesting that its role is closely related to CS recognition rather than association with the drugs. Finally, these data revealed that constricting and flexing specific regions in PR while allowing flexible movement of the substrate can allow for multiple, complex mechanisms of resistance to occur.
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